The enhanced safety, stability, and accelerated product development generally provided by DNA vaccination make it an appealing approach to develop a "universal" influenza vaccine. Unfortunately, successful DNA vaccination of primates requires multiple inoculations with undesirably large doses of plasmid. Genetically encoded adjuvants could provide the dose-sparing effect necessary to realize a practical DNA-based "universal" vaccine for influenza. We intend to exploit dominant-positive versions of Toll-like receptors (DP-TLRs) to provide the immune activation necessary to initiate such a protective immune response from DNA vaccination. Our vaccine targets for these "proof-of-concept" studies will be matrix 2 (M2) and nucleoprotein (NP), two antigens that exhibit a high degree of homology among strains of influenza and have been widely considered for such "universal" vaccines. We will pursue our objectives through the following specific aims: (1) construct DP-versions of TLR2, TLR4 and TLR9 and evaluate their capacity to activate NF-?B in human and mouse in vitro cell based assays; (2) evaluate the adjuvanticity of DP-TLRs co-formulated with plasmids expressing influenza M2 and NP in mice and (3) evaluate the dose-sparing effects of the DP- TLRs for the plasmids expressing influenza M2 and NP in murine viral challenge study. The adjuvant constructs that significantly improve immunogenicity or yield significant reductions in the protective dose over GM-CSF or IL-12 will be further evaluated in mice, ferret, and primate studies as components of an advanced influenza DNA vaccine in a Phase II SBIR application. PUBLIC HEALTH RELEVANCE: The objective of this project is to develop novel adjuvants that will support and improve the efficacy of a "universal" influenza DNA vaccine. These adjuvants will be based on dominant-positive toll-like receptors that activate through NF-?B signalling. [unreadable] [unreadable] [unreadable]